Thursday, 23 October 2014

As we've written about before,
mitochondria generate the energy required by our cells through respiration that
involves using an "electrochemical gradient" as an energy store (a
bit like pumping water up into a reservoir for energy storage to then harness it
flowing down the gradient of a hill to turn a turbine), and produces superoxide (free
oxygen radicals) as a by-product (a bit like sparks when the pumps are running
hot). The fundamental importance of this machinery which not only delivers
energy, but is also involved in disease and aging has led to its investigation in great molecular
detail (comparable to taking the turbines and generators apart to learn about
their function). Much less is known about how mitochondria actually behave when
they are fully functional in their natural environment inside our cells
(comparable to looking at the fully intact and running turbine), and progress
has been difficult since suitable `tools' are scarce.

A debate exists in the scientific literature about one of the key "tools"
used in the investigation of living cells. A particular fluorescent sensor protein
called cpYFP (circularly permuted yellow fluorescent protein) is used in
biological experiments, ostensibly as a way of measuring the levels of superoxide/free
oxygen radicals in a mitochondrion. Our
colleagues, however, have cast doubt on the ability of cpYFP to measure superoxide, providing evidence that it instead responds to pH,
part of the above electrochemical gradient. This debate was complicated by the
fact that in biology, pH and superoxide can vary together,
as the amount of "driving" and amount of "sparks" might be
expected to.

As another analogy: If we found an unknown
measuring device and we did not know how it works, but we saw that it responds
during sunny weather, we may conclude that it measures warm temperature.
However, it may in fact measure high atmospheric pressure which is, like warm
temperatures, often correlated with good weather.

The protein cpYFP changes its fluorescence in response to pH changes, but is unaffected by superoxide changes.

A recent and fascinating paper in Nature observed
that "flashes" of the cpYFP sensor during early development of worms (as
a model for other animals and humans) were correlated with their eventual
lifespan. However, despite the debate about what it is exactly that the cpYFP sensor measures, the paper interpreted
it as responding to superoxide: looking at the
correlation in the light of the so called “free radical theory of aging". This long-standing
and much debated theory hypothesizes that the cause of why we age and
eventually die is related to the constant production of free oxygen radicals in
our mitochondria causing a steady increase in damage to our cells weakening
their energetic machinery more and more and making them
prone to illnesses.

In response to this, our colleagues decided to settle the question about what
the sensor actually measures chemically, removing biological complications from
the system. In the analogy of the unknown measurement device, the device was
now tested under controlled temperature and controlled pressure to clearly distinguish
between the two. They produced an experimental setup where a mix of chemicals
was used to generate superoxide in the absence of any pH
change. cpYFP in this mix did not show any signal, showing that it remains
unresponsive to superoxide. In concert, they showed that
even small changes in pH produced a dramatic response in cpYFP signal. Finally,
they investigated the physical structure of cpYFP, showing that a large opening
in the barrel-like structure of the protein exposes a pH-sensitive chemical
group to its environment (comparable to showing how exactly the inner mechanics
of the unknown measurement device can pick up pressure changes). We thus
concluded, in a recent publication "The ‘mitoflash’ probe cpYFP does not respond to superoxide" (in the journal Nature here) that the cpYFP sensor reports pH
rather than superoxide, and that results using cpYFP
(including the above Nature paper, which remains fascinating) should be
interpreted as such. Iain, Markus and Nick